The present invention relates to a novel process for preparing optically active 3,3,3-trifluoro-2-alkylpropionic acid derivatives of the general formulae 
and to novel representatives of this compound class.
Up until now, the known 3,3,3-trifluoro-2-alkylpropionic acid derivatives, such as 3,3,3-trifluoro-2-amino-2-methylpropionic acid, have been synthesized using an amino acylase and starting from racemic trifluoracetyl-R(+)2-trifluoromethylalanine. The product is only formed in low yield in this method.
3,3,3-Trifluoro-2-alkylpropionic acid derivatives, such as 3,3,3-trifluoro-2-hydroxy-2-ethylpropionic acid, are important intermediates for preparing therapeutic amides (EP-A 0 524 781).
Up until now, the known 3,3,3-trifluoromethyl-2-alkylpropionic acid derivatives, such as 3,3,3-trifluoro-2-amino-2-methylpropionic acid, have been synthesized using an amino acylase and starting from racemic trifluoroacetyl-R(+)2-trifluoromethylalanine. The product is only formed in low yield in this method.
The object of the present invention was to make available a process for preparing optically active 3,3,3-trifluoro-2-alkylpropionic acid derivatives in which the desired products are formed both in excellent yield and in relatively short reaction times.
This object is achieved by the process as claimed in claim 1.
According to the invention, the 3,3,3-trifluoro-2-alkylpropionic acid derivatives are prepared by transforming a racemic 3,3,3-trifluoro-2-alkylpropionamide of the formula 
in which R denotes ethyl or methyl and X denotes xe2x80x94OH or NH2, with the exception that, when R=methyl, Xxe2x89xa0OH, either using microorganisms which are capable of utilizing the latter, as their sole source of nitrogen, in the form of the racemate or of one of its optically active isomers, or using a polypeptide which possesses amidohydrolase activity and which is capable of hydrolyzing the latter.
The starting compounds, i.e. the 3,3,3-trifluoro-2-alkylpropionamides of the general formula III, can be prepared using customary chemical methods. The starting compounds, such as 3,3,3-trifluoro-2-hydroxy-2-ethylpropionamide, are preferably prepared from the corresponding 3,3,3-trifluoro-2-alkylpropiononitrile of the general formula 
in which R and X have said meaning, by hydrolyzing it with a mineral acid.
The mineral acid which is used can be one which is customary for the skilled person, for example phosphoric acid, hydrochloric acid or sulfuric acid. The mineral acid which is used is preferably sulfuric acid. The mineral acid is expediently employed in excess, preferably in a quantity of from 2 to 4 mol per mole of propiononitrile.
The hydrolysis is expediently carried out at a temperature of from 20 to 140xc2x0 C., preferably of from 90 to 120xc2x0 C.
All the microorganisms which are capable of utilizing, as their sole source of nitrogen, the propionamide of the formula III in the form of the racemate or of one of its optically active isomers, and also all the isolated polypeptides which possess amidohydrolase activity and which are capable of hydrolyzing the propionamide of the formula III, can be used for the biotransformation according to the invention.
Microorganisms, and polypeptides having amidohydrolase activity, which possess this property, and also the isolated and sequenced DNA fragment which encodes the amidohydrolase are already known and described in WO 98/01568. The microorganisms which can be employed are both what are termed the xe2x80x9cwild-type strainsxe2x80x9d, which can be isolated from soil samples, sludge or effluent, as described in WO 98/01568, or what are termed xe2x80x9crecombinantly altered microorganismsxe2x80x9d, which are transformed with the isolated DNA fragment, as described in the WO. The biotransformation is expediently carried out using microorganisms of the genus Klebsiella or using xe2x80x9crecombinantly altered microorganismsxe2x80x9d. The microorganisms of the genus Klebsiella which are preferably employed are those of the species Klebsiella oxytoca PRSI (DSM 11009), Klebsiella oxytoca PRSIK17 (DSM 11623), Klebsiella panticula ID-624 (DSM 11354) or Klebsiella pneumoniae ID-625 (DSM 11355) or their functionally equivalent variants and mutants. Examples of suitable xe2x80x9crecombinantly altered microorganismsxe2x80x9d are microorganisms of the species Escherichia coli DH5 and Escherichia coli XL-1 Blue MRF, in each case harboring plasmid pPRS1b, pPRS2a (DSM 11635), pPRS4 or pPRS7. Preference is given to using Escherichia coli XL-1 Blue MRF harboring plasmid pPRS7. The microorganisms having the designation DSM 11009 were deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen [German Collection of Microorganisms and Cell Cultures] GmbH, Mascheroderweg 1b, D-38124 Braunschweig, in accordance with the Budapest Treaty, on Jun. 24, 1996, while those having the designation DSM 11623 were deposited in this same institution, and in accordance with the Budapest Treaty, on Jun. 20, 1997, those having the designations DSM 11354 and DSM 11355 on Dec. 27, 1996 and those having the designation DSM 11635 on Jun. 30, 1997.
The biotransformation can be carried out using resting cells (cells which are not growing and which no longer require any C source or energy source) or using growing cells, after the microorganisms have been cultured in a customary manner. The biotransformation is preferably carried out using resting cells.
It is possible to employ media which are customary to the skilled person, such as low molecular weight phosphate buffer, HEPES buffer or mineral salt media, for the biotransformation.
The biotransformation is expediently carried out while adding the 3,3,3-trifluoro-2-alkylpropionamide of the formula III on one occasion or continuously such that its concentration does not exceed 10% by weight, preferably 2.5% by weight.
The pH of the medium can be in a range of from 4 to 12, preferably of from 5 to 11. Expediently, the biotransformation is carried out at a temperature of from 10 to 80xc2x0 C., preferably of from 10 to 50xc2x0 C.
After a customary transformation time of from 1 min to several days, the optically active 3,3,3-trifluoro-2-alkylpropionic acid derivatives of the general formulae I and II can then be isolated using customary working-up methods, such as extraction.
It was found that, when 3,3,3-trifluoro-2-amino-2-methylpropionamide is employed as the substrate, the corresponding R-acid is formed within 5 minutes and the corresponding S-amide can then be isolated.
The racemic 3,3,3-trifluoro-2-hydroxy-2-ethylpropionamide of the general formula III and the corresponding (+)-acid, the corresponding (xe2x88x92)-amide and the (S)-3,3,3-trifluoro-2-amino-2-methylpropionamide are all compounds which have not previously been described in the literature and consequently form part of the invention.
It was furthermore found that the optically active 3,3,3-trifluoro-2-alkylpropionamides of the general formula I can be hydrolyzed, in a known manner, into the optically active 3,3,3-trifluoro-2-alkylpropionic acid derivatives of the general formula II. In this connection, preference is given to hydrolyzing (S)-3,3,3-trifluoro-2-amino-2-methylpropionamide into (S)-3,3,3-trifluoromethyl-2-amino-2-methylpropionic acid.
The hydrolysis is effected in analogy with the hydrolysis described in WO 98/01568. This hydrolysis preferably takes place either chemically, in the presence of a base, or microbiologically, using microorganisms of the genus Rhodococcus.